Causes of a low platelet count generally fall under one of the several categories

Reduced production of platelets

Platelets are produced in your bone marrow. A medical problem that involves your bone marrow, such as occurs with leukemia and some types of anemia, could lead to a reduction in the number of new platelets produced. Viral infections, including HIV infection, may suppress your bone marrow's ability to make platelets. Other cancers that affect bone marrow, chemotherapy drugs and heavy alcohol consumption also can impair platelet production.

Increased breakdown of platelets

A number of conditions can cause your body to use up or destroy platelets more rapidly than they are produced, leading to a shortage of platelets in your bloodstream. Examples include:

 Pregnancy, which may cause mild thrombocytopenia.

 Idiopathic thrombocytopenic purpura (ITP), a condition in which your immune system mistakenly identifies platelets as a threat and forms antibodies that attack them.

 Other autoimmune diseases, such as lupus and rheumatoid arthritis, which may lead to destruction of platelets due to a malfunctioning immune system.

 Blood poisoning (septicemia) from severe bacterial infections, which may lead to destruction of platelets.

 Thrombotic thrombocytopenic purpura (TTP), a rare, life-threatening condition that occurs when small blood clots suddenly form throughout your body, using up large numbers of platelets. TTP can happen sporadically or as a side effect of some medications.

 Hemolytic uremic syndrome, another rare disorder that causes a sharp drop in platelets, destruction of red blood cells and impairment of kidney function. Sometimes, this can occur in association with a bacterial Escherichia coli (E. coli) infection, such as may be acquired from eating raw or under*****d meat (often hamburger).

Sometimes, certain medications can cause a thrombocytopenic reaction by confusing the immune system and causing it to destroy platelets. Examples include heparin, quinidine, quinine, sulfa-containing antibiotics, some oral diabetes drugs, gold salts and rifampin. Sometimes, heparin-induced thrombocytopenia can cause excessive blood clotting instead of bleeding, increasing the risk of clot formation deep within a leg blood vessel or the transport of such a clot to your lungs, which can be life-threatening.

Trapping of platelets in the spleen

The spleen is a small organ about the size of your fist located just below your rib cage on your left side. Normally, your spleen works to fight infection and filter unwanted material from your blood. An enlarged spleen — which can be caused by a number of disorders — may harbor too many platelets, causing a decrease in the number of platelets in circulation.

A dysmorphic neonate with mild to moderate thrombocytopenia (50,000-150,000/mcL) most likely has a genetic syndrome associated with thrombocytopenia, such as trisomy 13, 18, 21, triploidy, or Turner's, Noonan's, Alport, or Wiskott-Aldrich syndromes. Some ****bolic disorders, such as the organic acidemias isovaleric and methylmalonic acidemia, are also associated with neonatal thrombocytopenia.

Thrombocytopenic absent radius syndrome (TARS) is an autosomal recessive disorder in which radii are absent bilaterally but thumbs are present. Forearms are foreshortened and bowed. Thrombocytopenia is severe, and a third of babies also have congenital heart disease. Platelet counts of infants with TARS gradually decrease over the first year of life, resulting in early death from bleeding at 1 or 2 years of age.

Congenital amegakaryocytic thrombocytopenia is caused by a defect in the Tpo receptor. Severe thrombocytopenia with eventual pancytopenia usually leads to death from hemorrhage.

PrincipleWhole blood is diluted with a 1% ammonium oxalate solution. The isotonic balance of thediluent is such that all erythrocytes are lysed while the leukocytes, platelets, and reticulocytesremain intact.1,2The standard dilution for platelet counts is 1:100. This dilution is prepared usingthe leukocyte/platelet Unopette system.1The dilution is mixed well and incubated to permit lysisof the erythrocytes. Following the incubation period, the dilution is mounted on ahemacytometer. The cells are allowed to settle and then are counted in a specific area of thehemacytometer chamber under the microscope. The number of platelets is calculated per µL (x109/L) of blood.Reagents and Equipment1.Two leukocyte/platelet Unopette reservoirs; each containing 1.98 mL of the following diluent:Ammonium oxalate11.45 gSorensen’s phosphate buffer 1.0 gThimerosal0.1 gQS with distilled water to 1 liter2.Two Unopette capillary pipets, 20 µL3.Hemacytometer with cover glass4.Petri dish with filter paper5.Hand counter6. MicroscopeQuality ControlCommercial quality control materials with established control limits should be run periodically.The frequency is determined by each laboratory's workload. For instance, quality controlmaterial may be run at the beginning of each eight-hour shift.SpecimenWhole blood, anticoagulated with EDTA, or free-flowing capillary blood may be used.
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Procedure1.Prepare two leukocyte/platelet Unopettes as follows:a. Using the protective shield on the capillary pipet, puncture the diaphragm asfollows:1)Place reservoir on a flat surface. Grasping the reservoir in one hand, takethe pipet assembly in the other hand and push the tip of the pipet shieldfirmly through the diaphragm in the neck of the reservoir, then remove.b. Remove the shield from the pipet assembly with a twist and fill the capillary pipetwith whole blood. Transfer the whole blood to reservoir as follows:1)Wipe excess blood from the outside of the capillary pipet, making certainthat no blood is removed from the capillary bore.2)Squeeze the reservoir slightly to force out some air. Maintain pressure onthe reservoir.3)Cover opening of overflow chamber of the pipet with your index fingerand seat the pipet securely in the reservoir neck.4)Release pressure on the reservoir. Then remove your finger from the pipetopening. Negative pressure will draw the blood into the diluent.5)Squeeze the reservoir gently two or three times to rinse the capillary bore,forcing diluent into, but not out of, the overflow chamber, releasing pressureeach time to return the mixture to the reservoir.6)Place your index finger over the pipet opening and gently invert severaltimes to thoroughly mix the blood with diluent. 7)Let stand for 10 minutes to allow erythrocytes to hemolyze.2.Clean the hemacytometer and cover glass by flooding them with 70% alcohol. Drythoroughly with gauze or tissue; do not allow the alcohol to dry on the hemacytometer.Be sure to remove all lint. Place the cover glass in position over the ruled area.3. Following incubation, mix diluted blood thoroughly by inverting reservoir to resuspendcells. Charge hemacytometer as follows:a.Convert to dropper assembly by withdrawing the pipet from the reservoir andreseating it securely in its reverse position.b.Clean the capillary bore by inverting the reservoir, gently squeeze the sides, anddiscard the first three or four drops.
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c.Place the pipet tip on the edge of the ruled area of the counting chamber.Carefully charge the hemacytometer with diluted blood by gently squeezing thesides of the reservoir to expel the contents until the chamber is properly filled.d.Repeat procedure to charge the other side of the hemacytometer with the firstUnopette reservoir.e.Place the hemacytometer on moistened filter paper in a Petri dish, and allow tostand 10 minutes to permit the cells to settle.f.Using this same procedure, charge a second hemacytometer with the second Unopette reservoir.4.Carefully place the hemacytometer on the microscope stage. Perform cell count asfollows:a. With the low-power (10x) objective, locate the ruled area and the center largesquare (1 mm2). Examine the entire center square for even distribution ofplatelets, then carefully switch to the high-dry-power (40x) phase objective forcounting platelets. With phase microscopy, platelets appear as round or ovalbodies.b.Platelets are counted in the entire center large square (1 mm2) (Figure 7-7) asfollows:1)Count the platelets in the first row of squares going from left to right, thenfrom right to left in the second row; follow this pattern until all rows arecounted.2)Within each square, count all platelets touching the top and left-handborders. Do not count any cells touching the bottom or right-hand borders.3)Use the fine adjustment knob to focus up and down to identify theplatelets.c.Repeat this counting procedure for the other side of the hemacytometer.d.Record the counts for each center square. The difference between these twocounts should not exceed 10%.e.Count the platelets on the second hemacytometer following the above countingprocedure.Calculations
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1. The calculation formula for hemacytometer cell counts determines the number of cellswithin 1 µL (1 mm3) of blood (Figure 7-9). To make this determination, the total numberof cells counted must be corrected for the initial dilution of blood and the volume ofdiluted blood used. The standard dilution of blood for platelet counts is 1:100; thereforethe dilution factor is 100. The volume of diluted blood used is based on the area anddepth of the counting area. The area counted is 2 mm2 and the depth is 0.1 mm; thereforethe volume factor is 0.2 mm3 .Total number of cells counted • dilution factor • 1/volume factor = cells/mm3Cells/mm3= cells/µL or cells/µL • 103µL /L = cells x 109/LExample: 100 x 103platelets/µL • 103µL/L = 100 x 109platelets/L2.Average the platelet counts from the duplicate pipets and report result (x 109/L or /mm3).Reference Interval150-440 x 109 /LComments1.Platelet counts should be performed within three hours after the dilution has been prepared.2.The coefficient of variation (CV) for the 95% confidence limits is + 22%.3. If blood is collected by a skin puncture, carefully remove the first drop of blood andcollect free-flowing blood for the platelet count. This will minimize the occurrence ofplatelet clumping and adhesion of platelets to the puncture site.4.If clumps of platelets are seen in the hemacytometer, the procedure should be repeated.Clumps may be due to inadequate mixing of blood or to poor technique in obtaining theblood specimen.5.A Wright's-stained peripheral blood smear should be examined and the platelet estimatedetermined to confirm the hemacytometer platelet count. The platelet estimate shouldcorrelate with the platelet count + 25%. If a discrepancy exists, the platelet count andperipheral blood smear estimate should be repeated.6.In acute leukemia, there is an increase of blast cells in the peripheral blood. Oftenfragments of cytoplasm about the size of platelets break off the blast cells. Thesecytoplasmic fragments are called hyaline bodies. They are the same size and density asplatelets and may be counted as platelets by the hemacytometer or automated methods. It
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is very important that all platelet counts be confirmed by slide examination so that a falseincrease of platelets (due to the counting of fragments) is not reported.7.Platelet satellitism will result in falsely decreased platelet counts. With plateletsatellitism, platelets adhere to neutrophils when the blood sample is anticoagulated withEDTA and is not free to be counted. Platelet satellitism can be corrected by redrawing theblood sample using sodium citrate as the anticoagulant. The resulting platelet count mustbe multiplied by 1.1 to account for the dilutional effect of the citrate anticoagulant.8.A second method for manual platelet counts is the Rees Ecker method. Using theerythrocyte diluting pipet, whole blood is diluted with a solution containing brilliantcresyl blue, which stains the platelets a light bluish color. The platelets are then countedusing a standard hemacytometer and bright field microscopy.9.Technical sources of error in hemacytometer cell counts are given in Web Table 7-4.References1.Becton-Dickinson. Unopette WBC/Platelet determination for manual methods.Rutherford, N.J.: Becton, Dickinson, and Company; 1996.2.Brecher G, Cronkite EP. Morphology and enumeration of human blood platelets. J ApplPhysiol. 1950; 3:365.Copyright ' 1995-2003 by A Pearson Company Prentice-Hall, Inc. Legal Notice